This invention relates to methods for cycle sequencing.
Cycle sequencing is a procedure by which the nucleotide base sequence of a nucleic acid molecule is determined. The method involves repeatedly providing a DNA sequencing primer in the presence of deoxynucleoside triphosphates and one or more dideoxynucleoside triphosphates in a suitable buffer solution with the template DNA to be sequenced. Denaturation of the extended primer from the template DNA is achieved by periodic heating after periods at lower temperatures at which primer extension occurs. Generally, this procedure is carried out using a thermostable enzyme, such as Thermus aquaticus DNA polymerase (Taq), so that more polymerase need not be added after each heating step. That is, when the cycling is between a temperature of 37.degree. C. and 95.degree. C. non-thermostable enzymes, such as T7 DNA polymerase or Klenow fragment of DNA polymerase I are denatured, and must be added after each heating step at 95.degree. C. In contrast, Taq is stable to heating at 95.degree. C. and thus, requires no additional enzyme to be added at each cycle.